An alternative approach to study the enzymatic specificities of the CfrBI restriction-modification system.

Restriction-modification systems (RMS) are the main gene-engineering tools and a suitable model to study the molecular mechanisms of catalysis and DNA-protein interactions. Research into the catalytic properties of these enzymes, determination of hydrolysis and DNA-methylation sites remain topical. In our previous work we have cloned and sequenced the CfrBI restriction-modification system (strain Citrobacter freundii), which recognizes the nucleotide sequence 5'-CCWWGG-3'. In this article we describe the cloning of the methyltransferase and restriction endonuclease genes (gene encoding CfrBI DNA methyltransferase (cfrBIM) and gene encoding CfrBI restriction endonuclease (cfrBIR)) separately to obtain strains overproducing the enzymes of this system. His6-CfrBI, which had been purified to homogeneity, was used to establish the DNA-hydrolysis point in its recognition site. CfrBI was shown to cleave DNA after just the first 5'C within the recognition site and then to generate 4-nt 3' cohesive ends (5'-C/CWWGG-3'). To map the site of methylation by M.CfrBI, we exploited the fact that the CfrBI site partially overlaps with the recognition sites of the well-documented enzymes KpnI and ApaI. The M.CfrBI- induced hemimethylation of the internal C residue of the ApaI recognition sequence (GGGCN4mCC) was observed to block cleavage by ApaI. In contrast, KpnI was able to digest its M.CfrBI-hemimethylated site (GGTAN4mCC). KpnI was used to restrict a fragment of DNA harbouring the CfrBI and KpnI sites, in which the CfrBI site was methylated in vitro by His6-M.CfrBI using [3H]-SAM. The subsequent separation of hydrolysis products by electrophoresis and the enumeration of incorporated [H3]-methyl groups in each of the fragments made it possible to determine that external cytosine undergoes modification in the recognition site.

Type II restriction endonuclease R.Eco29kI is a member of the GIY-YIG nuclease superfamily.

BACKGROUND: The majority of experimentally determined crystal structures of Type II restriction endonucleases (REases) exhibit a common PD-(D/E)XK fold. Crystal structures have been also determined for single representatives of two other folds: PLD (R.BfiI) and half-pipe (R.PabI), and bioinformatics analyses supported by mutagenesis suggested that some REases belong to the HNH fold. Our previous bioinformatic analysis suggested that REase R.Eco29kI shares sequence similarities with one more unrelated nuclease superfamily, GIY-YIG, however so far no experimental data were available to support this prediction. The determination of a crystal structure of the GIY-YIG domain of homing endonuclease I-TevI provided a template for modeling of R.Eco29kI and prompted us to validate the model experimentally. RESULTS: Using protein fold-recognition methods we generated a new alignment between R.Eco29kI and I-TevI, which suggested a reassignment of one of the putative catalytic residues. A theoretical model of R.Eco29kI was constructed to illustrate its predicted three-dimensional fold and organization of the active site, comprising amino acid residues Y49, Y76, R104, H108, E142, and N154. A series of mutants was constructed to generate amino acid substitutions of selected residues (Y49A, R104A, H108F, E142A and N154L) and the mutant proteins were examined for their ability to bind the DNA containing the Eco29kI site 5'-CCGCGG-3' and to catalyze the cleavage reaction. Experimental data reveal that residues Y49, R104, E142, H108, and N154 are important for the nuclease activity of R.Eco29kI, while H108 and N154 are also important for specific DNA binding by this enzyme. CONCLUSION: Substitutions of residues Y49, R104, H108, E142 and N154 predicted by the model to be a part of the active site lead to mutant proteins with strong defects in the REase activity. These results are in very good agreement with the structural model presented in this work and with our prediction that R.Eco29kI belongs to the GIY-YIG superfamily of nucleases. Our study provides the first experimental evidence for a Type IIP REase that does not belong to the PD-(D/E)XK or HNH superfamilies of nucleases, and is instead a member of the unrelated GIY-YIG superfamily.

Protein NCRII-18: the role of gene fusion in the molecular evolution of restriction endonucleases.

This work first constructed the fusion protein NCRII-18 by fusing the restriction endonuclease Ecl18kI gene and part of the gene coding for the N-terminal domain of the endonuclease EcoRII. The fusion of the EcoRII N-terminal domain leads to a change in the properties of the recombinant protein. Unlike Ecl18kI, which made the basis of NCRII-18, the fusion protein predominantly recognizes the CCWGG sites, having lost the capability of interacting with the CCSGG sites. Experimental data support the hypothesis of a close evolutionary relationship between type IIE and IIP restriction endonucleases via a recombination between domains with active site structure and elements for recognition with domains responsible for recognition of DNA sequences.

Protein SgpR of Pseudomonas putida strain AK5 is a LysR-type regulator of salicylate degradation through gentisate.

Pseudomonas putida strain AK5 was the first characterized natural strain containing the 'classical' nah1 operon and nahR gene along with genes whose products are responsible for the less explored pathway of salicylate degradation through gentisate (the sgp operon). The sgp operon was found to be preceded by the divergently directed sgpR gene. The amino acid sequence of the sgpR product qualifies it as a LysR-type transcriptional regulator (LTTR) and suggests its potential function as an sgp operon transcriptional regulator. This study focused on verification of SgpR's involvement in regulation of transcription of the operon genes and characterization of its interaction with the sgp promoter. We determined the transcription start for sgpAIKGHB and identified the SgpR binding site. The equilibrium dissociation constant (KD) of the SgpR-DNA complex determined in the presence and absence of the inducer salicylate appeared to be, on the whole, at the lower end of the range for KD values reported for LTTRs. RT-qPCR showed that in the presence of salicylate, efficiency of transcription of the sgpAIKGHB operon increased by three orders of magnitude and reached the highest values so far observed for LTTR-controlled operons, thus holding much promise for further studies of the mechanism of transcriptional regulation that involves SgpR.

Xylanases of Cellulomonas flavigena: expression, biochemical characterization, and biotechnological potential.

Four xylanases of Cellulomonas flavigena were cloned, expressed in Escherichia coli and purified. Three enzymes (CFXyl1, CFXyl2, and CFXyl4) were from the GH10 family, while CFXyl3 was from the GH11 family. The enzymes possessed moderate temperature stability and a neutral pH optimum. The enzymes were more stable at alkaline pH values. CFXyl1 and CFXyl2 hydrolyzed xylan to form xylobiose, xylotriose, xylohexaose, xylopentaose, and xylose, which is typical for GH10. CFXyl3 (GH11) and CFXyl4 (GH10) formed the same xylooligosaccharides, but xylose was formed in small amounts. The xylanases made efficient saccharification of rye, wheat and oat, common components of animal feed, which indicates their high biotechnological potential.

Recent Origin of the Methacrylate Redox System in Geobacter sulfurreducens AM-1 through Horizontal Gene Transfer.

The origin and evolution of novel biochemical functions remains one of the key questions in molecular evolution. We study recently emerged methacrylate reductase function that is thought to have emerged in the last century and reported in Geobacter sulfurreducens strain AM-1. We report the sequence and study the evolution of the operon coding for the flavin-containing methacrylate reductase (Mrd) and tetraheme cytochrome с (Mcc) in the genome of G. sulfurreducens AM-1. Different types of signal peptides in functionally interlinked proteins Mrd and Mcc suggest a possible complex mechanism of biogenesis for chromoproteids of the methacrylate redox system. The homologs of the Mrd and Mcc sequence found in δ-Proteobacteria and Deferribacteres are also organized into an operon and their phylogenetic distribution suggested that these two genes tend to be horizontally transferred together. Specifically, the mrd and mcc genes from G. sulfurreducens AM-1 are not monophyletic with any of the homologs found in other Geobacter genomes. The acquisition of methacrylate reductase function by G. sulfurreducens AM-1 appears linked to a horizontal gene transfer event. However, the new function of the products of mrd and mcc may have evolved either prior or subsequent to their acquisition by G. sulfurreducens AM-1.

Oligomeric structure diversity within the GIY-YIG nuclease family.

The GIY-YIG nuclease domain has been identified in homing endonucleases, DNA repair and recombination enzymes, and restriction endonucleases. The Type II restriction enzyme Eco29kI belongs to the GIY-YIG nuclease superfamily and, like most of other family members, including the homing endonuclease I-TevI, is a monomer. It recognizes the palindromic sequence 5'-CCGC/GG-3' ("/" marks the cleavage position) and cuts it to generate 3'-staggered ends. The Eco29kI monomer, which contains a single active site, either has to nick sequentially individual DNA strands or has to form dimers or even higher-order oligomers upon DNA binding to make a double-strand break at its target site. Here, we provide experimental evidence that Eco29kI monomers dimerize on a single cognate DNA molecule forming the catalytically active complex. The mechanism described here for Eco29kI differs from that of Cfr42I isoschisomer, which also belongs to the GIY-YIG family but is functional as a tetramer. This novel mechanism may have implications for the function of homing endonucleases and other enzymes of the GIY-YIG family.